Optimizing Contrast in Automated 4D STEM Cryotomography.

4D STEM is an emerging approach to electron microscopy. While it was developed principally for high-resolution studies in materials science, the possibility to collect the entire transmitted flux makes it attractive for cryomicroscopy in application to life science and radiation-sensitive materials where dose efficiency is of utmost importance. We present a workflow to acquire tomographic tilt series of 4D STEM data sets using a segmented diode and an ultrafast pixelated detector, demonstrating the methods using a specimen of a T4 bacteriophage. Full integration with the SerialEM platform conveniently provides all the tools for grid navigation and automation of the data collection. Scripts are provided to convert the raw data to mrc format files and further to generate a variety of modes representing both scattering and phase contrasts, including incoherent and annular bright field, integrated center of mass, and parallax decomposition of a simulated integrated differential phase contrast. Principal component analysis of virtual annular detectors proves particularly useful, and axial contrast is improved by 3D deconvolution with an optimized point spread function. Contrast optimization enables visualization of irregular features such as DNA strands and thin filaments of the phage tails, which would be lost upon averaging or imposition of an inappropriate symmetry.